Method of treating cancer using dithiocarbamate derivatives

ABSTRACT

Dithiocarbamate, particularly tetraethylthiuram disulfide, and thiocarbamate anions strongly inhibit the growth of cancer cells of a variety of cell types. Such inhibitory effect is enhanced by heavy metal ions such as copper ions, cytokines and ceruloplasmin. A method is presented for using tetraethylthiuram disulfide to reduce tumor growth, and to potentiate the effect of other anticancer agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/392,122, filed on Sep. 8, 1999 now U.S. Pat. No. 6,589,987,which is hereby incorporated herein in its entirety by reference whichclaims priority under 35 U.S.C. § 119(e) to Provisional U.S. ApplicationSer. No. 60/099,390, filed Sep. 8, 1998, now abandoned.

FIELD OF INVENTION

This invention generally relates to methods of treating cancer, andparticularly to methods of treating cancer using dithiocarbamatederivatives.

BACKGROUND OF THE INVENTION

Cancer, the uncontrolled growth of malignant cells, is a major healthproblem of the modern medical era and ranks second only to heart diseaseas a cause of death in the United States. While some malignancies, suchas adenocarcinoma of the breast and lymphomas such as Hodgkin's Disease,respond relatively well to current chemotherapeutic antineoplastic drugregimens, other cancers are poorly responsive to chemotherapy,especially non-small cell lung cancer and pancreatic, prostate and coloncancers. Even small cell cancer of the lung, initially chemotherapysensitive, tends to return after remission, with widespread metastaticspread leading to death of the patient. Thus, better treatmentapproaches are needed for this illness. Also, because almost allcurrently available antineoplastic agents have significant toxicities,such as bone marrow suppression, renal dysfunction, stomatitis,enteritis and hair loss.

The end of the twentieth century has seen a more dramatic increase inthe observed incidence of malignant melanoma than for all other types oftumors. The biology of malignant melanomas offers an example of theimportance of transcription factors for malignant cell propagation.Malignant melanomas have great propensity to metastasize and arenotoriously resistant to conventional cancer treatments such aschemotherapy and γ-irradiation. Development of malignant melanoma inhumans progresses through a multistage process, with transition frommelanocyte to nevi, to radial growth, and subsequently to the verticalgrowth, metastatic phenotype of autonomous melanomas, associated withdecreased dependence on growth factors, diminished anchorage dependence,reduced contact inhibition and increased radiation and drug resistance.

Much of the molecular understanding of melanoma progression has comefrom studying the response of cultured melanoma cells to mitogenicstimuli. In culture, melanocyte proliferation and differentiation arepositively regulated by agents that increase cAMP (See, P. M. Cox, etal., “An ATF/CREB binding motif is required for aberrant constitutiveexpression of the MHC class II Drα promoter and activation by SV40T-antigen,” Nucleic Acids Res. 20:4881-4887 (1992); R. Halaban, et al.,“Regulation of tyrosinase in human melanocytes grown in culture,” J.Cell Biol. 97:480-488 (1983); D. Jean, et al., “CREB and its associatedproteins act as survival factors for human melanoma cells,” J. Biol.Chem. 273:24884-24890 (1998); P. Klatt, et al., “Nitric oxide inhibitsc-Jun DNA binding by specifically targeted S-glutathionylation,” J.Biol. Chem. 274:15857-15864 (1999); J. M. Lehmann, et. al., “MUC18, amarker of tumor progression in human melanoma, shows sequence similarityto the neural cell adhesion molecules of the immunoglobulinsuperfamily,” Proc. Natl. Acad. Sci. U.S.A. 89:9891-9895 (1989); M.Luca, et al., “Direct correlation between MUC18 expression andmetastatic potential of human melanoma cells,” Melanoma Res.3:35-41(1993); J. P. Richards, et al., “Analysis of the structuralproperties of cAMP-responsive element-binding protein (CREB) andphosphorylated CREB,” J. Biol. Chem. 271:13716-13723 (1996); and S. Xie,et al., “Dominant-negative CREB inhibits tumor growth and metastasis ofhuman melanoma cells,” Oncogene 15:2069-2075 (1997)), and several cAMPresponsive transcription factors binding to CRE (the consensus motif5′-TGACGTCA-3′, or cAMP response element) play prominent roles inmediating melanoma growth and metastasis. In MeWo melanoma cells, thetranscription factor CREB (for CRE-binding protein) and its associatedfamily member ATF-1 promote tumor growth, metastases and survivalthrough CRE-dependent gene expression. See, D. Jean, et al., supra.Expression of the dominant negative KCREB construct in metastatic MeWomelanoma cells decreases their tumorigenicity and metastatic potentialin nude mice. See, S. Xie, et al., “Expression of MCA/MUC18 by humanmelanoma cells leads to increased tumor growth and metastasis,” CancerRes. 57:2295-2303 (1997). The KCREB-transfected cells display asignificant decrease in matrix metalloproteinase 2 (MPP2, the 72 kDacollagenase type IV) mRNA and activity, resulting in decreasedinvasiveness through the basement membrane, an important component ofmetastatic potential.

The cell surface adhesion molecule MCAM/MUC 18, which is involved inmetastasis, of melanoma (See, J. M. Lehmann, et al., supra; M. Luca, etal., supra; S. Xie, et al., supra), is also down-regulated by KCREBtransfection. See, S. Xie, et al., Cancer Res., supra. In addition,expression of KCREB in MeWo cells renders them susceptible tothapsigargin-induced apoptosis, suggesting that CREB and its associatedproteins act as survival factors for human melanoma cells, therebycontributing to the acquisition of the malignant phenotype. See, D.Jean, et al., supra.

Melanoma cells aberrantly express the major histocompatibility complexclass II (MHC II) antigens, normally found only in B-lymphocytes andantigen presenting cells of the monocyte/macrophage cell line. See, P.M. Cox, et al., “An ATF/CREB binding motif is required for aberrantconstitutive expression of the MHC class II Drα promoter and activationby SV40 T-antigen. Nucleic Acids Res.,” 20:4881-4887 (1992). In B₁₆melanoma cells this is due to activation of the MHC II Drα promoter byconstitutive activation of an ATF/CREB motif. CREB family proteins alsobind to the UV-response element (URE, 5′-TGACAACA-3′), and URE bindingof the CREB family member ATF2 confers resistance to irradiation and tothe chemotherapeutic drugs cis-platinum, 1-α-D-arabinofuranosylcytosine(araC) or mitomycin C in MeWo melanoma lines. See, Z. Ronai, et al.,“ATF2 confers radiation resistance to human melanoma cells,” Oncogene16:523-531 (1998)). Thus, CREB family transcription factors playimportant roles in the malignant potential of this important tumor type.This has led to the suggestion by others that targeted moleculardisruption of ATF/CREB-mediated transcription might be therapeuticallyuseful for controlling growth and metastases of relativelytreatment-resistant malignant melanoma. See, D. Jean, supra, and Z.Ronai, supra.

The positively charged DNA binding domain of many transcription factorscontains cysteines which can be oxidatively modified by agents such ashydrogen peroxide or nitric oxide (NO), stimulating repair processesthat result in formation of mixed disulfides between glutathione (GSH)and protein thiols. See, P. Klatt, et al., supra; and, H. Sies,“Glutathione and its role in cellular functions,” Free Rad. Biol. Med.27:916-921 (1999)). As a consequence of this so-called protein“S-glutathionylation”, the usually positively charged transcriptionfactor DNA binding domain develops an electronegative charge imparted bydual carboxylate end groups of GSH. The change in charge disruptstranscription factor binding to its respective DNA consensus sequence.See, P. Klatt, et al., supra and H. Sies, supra. This mechanism has beendemonstrated to explain how NO inhibits c-Jun DNA binding byspecifically targeted S-glutathionylation of cysteines within the DNAbinding region, and a similar mechanism has been suggested for hownitrosative stress in general might functionally inhibit the activity ofFos, ATF/CREB, Myb and Rel/NFκB family transcription factors. See, P.Klatt, et al., supra.

The dithiocarbamates comprise a broad class of molecules giving them theability to complex metals and react with sulfhydryl groups andglutathione. After metal-catalyzed conversion to their correspondingdisulfides, dithiocarbamates inhibit cysteine proteases by forming mixeddisulfides with critical protein thiols. See, C. S. I. Nobel, et al.,“Mechanism of dithiocarbamate inhibition of apoptosis: thiol oxidationby dithiocarbamate disulfides directly inhibits processing of thecaspase-3 proenzyme,” Chem. Res. Toxicol. 10:636-643 (1997). CREBcontains three cysteines in the DNA binding region (Cys³⁰⁰, Cys³¹⁰ andCys³³⁷) which are not essential for DNA binding but might providereactive sites for S-glutathionylation. See, S. Orrenius, et al.,“Dithiocarbamtes and the redox regulation of cell death,” Biochem. Soc.Trans. 24:1032-1038 (1996)).

Recently, dithiocarbamates containing a reduced sulfhydryl group, e.g.,pyrrolidinedithiocarbamate (PDTC) have been shown to inhibit theproliferation of cultured colorectal cancer cells. See, Chinery, et al.,“Antioxidants enhance the cytotoxicity of chemotherapeutic agents incolorectal cancer: a p53-independent induction of p21^(WAF1/CIP1) viaC/EBPβ,” Nature Med. 3:1233-1241 (1997); Chinery et al., “Antioxidantsreduce cyclooxygenase-2 expression, prostaglandin production, andproliferation in colorectal cancer cells.” Cancer Res. 58:2323-2327(1998).

In addition to their reduced thioacid form, dithiocarbamates exist inthree other forms, e.g., a) the disulfide, a condensed dimmer of thethioacid, with elimination of reduced sulfhydryl groups by disulfidebond formation; b) the negatively charged thiolate anion, generally asthe alkali metal salt, such as sodium; and c) the 1,1-dithiolatocomplexes of the transition elements, in which the two adjoining sulfuratoms of the dithiocarbamate are bound to the same titanium, vanadium,chromium, iron, cobalt, nickel, copper, silver or gold metal ion. Thedisulfide, thiolate anion and transition metal complexes ofdithiocarbamates are all structurally distinct from the reduced form ofPDTC used by Chinery, et al., in that they have no reduced sulfhydrylmolecular moiety and are incapable of functioning as antioxidants bydonating the proton from a reduced sulfhydryl to scavenge electrons offree radical species. Lacking a reduced sulfhydryl, thiocarbamatedisulfides, thiolate anions and transition metal complexes should,according to the teachings of Chinery, et al., have no activity asantiproliferative compounds against cancer, since these three nonreducedchemical forms of dithiocarbamates are incapable of functioning asantioxidants.

In U.S. patent application Ser. No. 09/392,122; filed Sep. 8, 1999, itwas reported that the dithiocarbamate disulfide disulfiram sensitizestumor cells to cancer chemotherapy and could be used in conjunction withcancer chemotherapeutic drugs to increase their effectiveness intreating neoplasms. Recently, this effect has been explained in work inwhich disulfiram was shown to prevent maturation of the P-glycoproteinpump, an ATP-driven 170-kd efflux pump on the plasma membrane that pumpsa variety of cytotoxic drugs out of cells. See, T. W. Loo, et al.,“Blockage of drug resistance in vitro by disulfiram, a drug used totreat alcoholism.” J. Natl. Cancer Inst. 92:898-902 (2000). This effectreduces P-glycoprotein-mediated drug resistance in tumor cells andsensitizes tumor cells to cancer chemotherapy.

It is therefore an object of the present invention to provide a methodfor the treatment of cancer.

Another object of the present invention is to provide pharmaceuticalcompositions for the treatment of cancer.

It is still another object of the present invention to provide arelatively less toxic agent available for use alone in combination withcurrent drugs in order to better treat cancer patients without riskinginjury from the therapy itself.

SUMMARY OF THE INVENTION

The present invention provides a method for treating established cancerusing dithiocarbamate disulfides, or thiocarbamate anions either alone,or in combination with a heavy metal ion, and thiocarbamate complexes ofheavy metal ions.

It has been discovered that dithiocarbamate disulfides and theircorresponding thiolate anions alone exhibit potent inhibitory effects ongrowth of established tumor cells in the absence of antioxidantsulfhydryl groups within their structure. Thiocarbamate disulfides andtheir corresponding thiolate anions are effective in inhibiting thegrowth of established melanomas and non-small cell lung cancer cells,which are known to be poorly responsive to currently availableneoplastic agents. In addition, it has further been surprisinglydiscovered that the antiproliferative and antineoplastic effect ofdithiocarbamate disulfides and their corresponding thiolate anions onestablished tumor cells is greatly potentiated by co-treatment of cancercells with a transitional metal salt in a concentration which by itselfdoes not impair cancer cell growth. The potentiating function of thetransition metal is to facilitate formation of the thiolate anion fromthe dithiocarbamate disulfide. Further the tumor cell growth inhibitioneffect can be significantly enhanced by the addition of heavy metal ionssuch as copper, zinc, gold and silver ion, as examples, or byadministering the thiocarbamate as a heavy metal ion complex.

The chemical activity of these species is not from antioxidant actionbut from stimulating formation of mixed disulfides between thedithiocarbamate and sulfhydryl moieties of cysteines located at criticalsites on cell proteins, such as the DNA binding region of transcriptionfactors needed to promote expression of gene products necessary formalignant cell proliferation.

Dithiocarbamates disulfides that are useful in the treatment of cancerinclude, but are not limited to, those of the formulas:

R₂R₃N(S)CS—SC(S)NR₂R₃

wherein R₁, R₂, R₃ and R₄ are the same or different and representhydrogen, and unsubstituted or substituted alkyl, akenyl, aryl, alkoxy,and heteroaryl groups. It is noted that the alkyl groups can includecycloalky and hetercycloalkyl groups. R₁, R₂ and the N atom in theformula can together form an N-heterocyclic ring, which is, e.g.,heterocycloalkyl or heterocycloaryl. Likewise, R₃, R₁ and the N atom inthe formula can together form an N-heterocyclic ring, which is, e.g.,heterocycloalkyl or heterocycloaryl. Typically R₁, and R₂ are not bothhydrogen, and R₃ and R₄ are not both hydrogen.

In accordance with another aspect of this invention, a method fortreating established cancer in a patient is provided comprisingadministering to the patient a therapeutically effective amount of adithiocarbamate disulfide, preferably disulfiram, or the correspondingdiethyldithiocarbamate thiolate anion, and a heavy metal ion of theformula:

wherein R₂ and R₃ are the same or different and represent hydrogen, andunsubstituted or substituted alkyl, akenyl, aryl, alkoxy, and heteroarylgroups; An is a metal, e.g., titanium, vanadium, chromium, iron, cobalt,nickel, copper, silver, silver or gold; n is the valence of the metal;and M is sodium, potassium, calcium, magnesium, barium, or lithium or ananion of small molecular weight.

In a preferred embodiment, the heavy metal ion is administered as acomplex or chelate with the dithiocarbamate disulfide or correspondingthiolate anion. Suitable heavy metal ions include but are not limited toions of arsenic, bismuth, cobalt, copper, chromium, gallium, gold, iron,manganese, nickel, silver, titanium, vanadium, selenium, and zinc.

In another preferred embodiment, the dithiocarbamate disulfide orcorresponding thiolate anion and the heavy metal ion are administered incombination with another anticancer agent.

In addition, the present invention provides a method for sensitizingcancer cells to chemotherapeutic drugs by the administration of adithiocarbamate thiolate anion or a dithiocarbamate complex with heavymetals in order to effect inhibition of the tumor cell membraneP-glycoprotein pump which functions to extrude from cancer cells theanti-neoplastic agents which are absorbed.

In accordance with another aspect of the invention, the presentinvention provides a pharmaceutical composition that comprises apharmaceutically acceptable carrier, and a complex between adithiocarbamate and a heavy metal ion. Optionally, the composition canfurther contain another anticancer agent.

The active compounds of this invention can be administered through avariety of administration routes. For example, they can be administeredorally, intravenously, intradermally, subcutaneously and topically.

The present invention is effective for treating various types of cancer,including but not limited to melanoma, non-small cell lung cancer, smallcell lung cancer, renal cancer, colorectal cancer, breast cancer,pancreatic cancer, gastric cancer, bladder cancer, ovarian cancer,uterine cancer, lymphoma, and prostate cancer. In particular the presentinvention will be especially effective in treating melanoma, lungcancer, breast cancer and prostate cancer. Thus the use ofdithiocarbamate disulfides and thiolate anions in this invention offersa readily available and easily used treatment for cancers in man andother animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows that M1619 melanoma cells exhibit constitutive DNA bindingactivity to the cyclic AMP response element (CRE);.

FIG. 1B shows that the thiocarbamate disulfide disulfiram and copperinhibit transcription factor binding to CRE;

FIG. 1C shows that EMSAs performed using nuclear protein from replicateexperiments (n=4) in which near confluent cells were treated for 8 hrwith FBS alone, DMSO vehicle (5 μl/well), disulfiram (5 μM), CuSO₄ (1.6μM), or the combination of disulfiram plus copper;

FIG. 2 shows the effect of adding disulfiram or disulfiram plus copperdirectly to binding reaction on transcription factor to DNA binding;

FIG. 3A shows disulfiram and copper reduce expression of the cell-cycleprotein cyclin A;

FIG. 3B replicates experiments (n=4 each) in which cells were treatedwith DMSO vehicle, (5 μl/ml, lanes 1-4), disulfiram (5 μM, lanes 5-8),(5 μl/ml), CuSO₄ (1.6 μM, lanes 9-12) or the combination of disulfiramand CuSO₄ (lanes 13-16). After 24 hours cells were lysed, immunoblotswere performed to assay for cyclin A;

FIG. 3C illustrates quantitation of experiments in FIG. 3B bydensitometry;

FIG. 4A shows that disulfiram inhibits proliferation of M1619 humanmelanoma, cell lines;

FIG. 4B illustrates that cell-impermeate Cu²⁺ chelatorbathocuproine-disulfonic acid prevents growth inhibition fromdisulfiram;

FIG. 4C sows supplementation of growth medium with copper enhances theantiproliferative activity of disulfiram;

FIG. 4D shows that ceruloplasmin can serve as a source of copper forenhancing the antiproliferative activity of disulfiram;

FIG. 5A shows M1619 melanoma cells treated with DMSO vehicle;

FIG. 5B shows M1619 melanoma cells treated with 5 μM disulfiram;

FIG. 6A shows that disulfiram combined with copper induces S-phase cellcycle arrest in M1619 melanoma cells and apopotosis;

FIG. 6B shows that 5 μM disulfiram combined with copper induces S-phasecell cycle arrest in M1619 melanoma cells and apopotosis;

FIG. 6C shows that 5 μM disulfiram plus 250 μg/ml ceruloplasmin (Cerulo)as a source of copper.

FIG. 7A shows that other metals also protentiate antiproliferativeactivity of disulfiram;

FIG. 7B shows the antiproliferative activity of disulfiram is enhancedby supplementation of medium with other heavy metals;

FIG. 7C shows complexes of disulfiram with gold demonstrate enhancedantiproliferative activity;

FIG. 7D shows the antiproliferative activity of the thiolate sodiumdiethyldithiocarbamate trihydrate (NaDDC) is reduced by lowconcentrations of DTT in the growth medium; and

FIG. 8 shows the X-ray crystallographic structure of complexes formedfrom mixing gold tetrachloride and disulfiram.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter withreference to the accompanying examples, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

As used herein, the term “dithiocarbamate disulfides” refers tocompounds having the formula of:

R₁R₂N(S)CS—SC(S)NR₃R₄

wherein R₁, R₂, R₃ and R₄ are the same or different and representhydrogen, and unsubstituted or substituted alkyl, akenyl, aryl, alkoxy,and heteroaryl groups. It is noted that the alkyl groups can includecycloalky and hetercycloalkyl groups. R₁, R₂ and the N atom in theformula can together form an N-heterocyclic ring, which is, e.g.,heterocycloalkyl or heterocycloaryl. Likewise, R₃, R₄ and the N atom inthe formula can together form an N-heterocyclic ring, which is, e.g.,heterocycloalkyl or heterocycloaryl. Typically R₁ and R₂ are not bothhydrogen, and R₃ and R₄ are not both hydrogen. Thus, dithiocarbamatedisulfide is a disulfide form of dithiocarbamates that have a reducedsulfhydryl group.

Many dithiocarbamates are known and synthesized in the art. Non limitingexamples of dithiocarbamates include diethyldithiocarbamate,pyrrolodinedithiocarbamate, N-methyl, N-ethyl dithiocarbamates,hexamethylenedithiocarbamate, imidazolinedithiocarbamates,dibenzyldithiocarbamate, dimethylenedithiocarbamate,dipolyldithiocarbamate, dibutyldithiocarbamate, diamyldithiocarbamate,N-methyl, N-cyclopropylmethyldithiocarbamate,cyclohexylamyldithiocarbamate, pentamethylenedithiocarbamate,dihydrxyethyldithiocarbamate, N-methylglucosamine dithiocarbamate, andsalts and derivatives thereof Typically, a sulfhydryl-containingdithiocarbamate can be oxidized to form a dithiocarbamate disulfide.

Sulfhydryl-containing dithocarbamates can be converted to theircorresponding thiolate anions by treatment with an alkali-metalhydroxide as a proton acceptor, yielding the structure:

wherein R₂ land R₃ are the same or different and represent hydrogen, andunsubstituted or substituted alkyl, akenyl, aryl, alkoxy, and heteroarylgroups; M is an alkali metal selected from the group consisting of fromthe group consisting of sodium, potassium, calcium, magnesium, barium,and lithium; and n is the valence of the alkali metal.

Finally, the heavy metal complexes of dithocarbamate can be synthesizedeither by treatment of the disulfide or the thiolate anion forms ofdithiocarbamates with metal salts, yielding a variety of useful metalcomplexes in which the metal forms a complex with both sulfur atoms:

wherein R₂ and R₃ are the same or different and represent hydrogen, andunsubstituted or substituted alkyl, akenyl, aryl, alkoxy, and heteroarylgroups; An is a metal, e.g., titanium, vanadium, chromium, iron, cobalt,nickel, copper, silver, silver or gold; n is the valence of the metal;and M is sodium, potassium, calcium, magnesium, barium, or lithium or ananion of small molecular weight.

Specifically, the preferred gold 1,1-dithio chelates of dithiocarbamateshas the formulae:

wherein R₂, R₃ are ethyl, and An is an anion of small molecular weight.

Any pharmaceutically acceptable form of dithiocarbamate disulfides,their corresponding thiolate anions and dithiocarbamate metal chelatescan be used. For example, tetraethylthiuram disulfide, which is known asdisulfiram, is used in one embodiment of this invention. Disulfiram hasthe following formula:

R₁R₂N(S)CS—SC(S)NR₃R₄

where R₁, R₂, R₃ and R₄ are all ethyl. Disulfiram has been usedclinically in the treatment of alcohol abuse, in which disulfiraminhibits hepatic aldehyde dehydrogenase.

The thiolate anion derivative of disulfiram is diethyldithiocarbamateanion, the sodium salt of which has the following formula:

Finally, the heavy metal complex of diethyldithiocarbamate, exemplifiedbelow as the gold (Au III) 1,1-dithiolato complex, is shown:

wherein R₂ and R₃are ethyl, and An is an anion of small molecularweight.

Methods of making dithiocarbamates and their disulfides are generallyknown in the art. Exemplary methods are disclosed in, e.g., Thorn, etal, The Dithiocarbamates and Related Compounds, Elsevier, N.Y., 1962;and U.S. Pat. Nos. 5,166,387, 4,144,272; 4,066,697, 1,782,111, and1,796,977, all of which are incorporated herein by reference.

The term “treating cancer” as used herein, specifically refers toadministering therapeutic agents to a patient diagnosed of cancer, i.e.,having established cancer in the patient, to inhibit the further growthor spread of the malignant cells in the cancerous tissue/ and/or tocause the death of malignant cells.

This invention provides a method for treating cancer in a patient. Inaccordance with the present invention, it has been discovered thatdithiocarbamate disulfides, their corresponding thiolate anions, andtheir heavy metal complexes, such as disulfiram, thediethyldithiocarbamate anion and dichloro(ditheylthiocarbamyl)gold (II),respectively, can inhibit the growth of tumor cells in a heavy metalion-dependent manner. Specifically, heavy metal ions such as copper,zinc, gold, and silver ions significantly enhance the inhibitory effectof dithiocarbamate disulfides and their thiolate anions on tumor cells,while depletion of such heavy metal ions prevents growth inhibition bydisulfiram and the diethyldithiocarbamate anion. The function performedby the metal is to chemically catalyze formation of or stabilize thethiolate anion form in vivo, so that the thiolate anion is able to formmixed disulfides with protein cysteine sulfhydryl groups of cellularproteins.

In accordance with one aspect of this invention, a method for treatingan established cancer in a patient is provided. A dithiocarbamatedisulfide can be administered to a patient having established cancer totreat the cancer. Preferably, the thiuram disulfide administered is atetra alkyl thiuram disulfide such as teraethylthiuram disulfide, i.e.,disulfiram.

In another aspect of the invention, the method for treating cancer in apatient comprises administering to the patient a therapeuticallyeffective amount of a dithiocarbamate thiolate anion.

In another aspect of the invention, the method for treating cancer in apatient comprises administering to the patient a therapeuticallyeffective amount of a dithiocarbamate disulfide or its thiolate anion,and a heavy metal ion.

Non-limiting examples of heavy metal ions include ions of arsenic,bismuth, cobalt, copper chromium, gallium, gold iron, manganese, nickel,silver, titanium, vanadium, selenium and zinc. Preferably, gold, silver,zinc, selenium, and copper ions are used. Sources of such heavy metalions are known to the those skilled in the art. For example, such ionscan be provided in a sulfate salt, or chloride salt form, or any otherpharmaceutically suitable forms. Preferably, the salt is in a chelatedform, complexed with a pharmaceutically acceptable organic anion such asacetate, glycinate, gluconate, propionate or lactate so that absorptionof the metal from the gastrointestinal tract is enhanced.

One or more dithocarbamate disulfide or corresponding thiolate anionsand one or more heavy metal ions can be administered to the patient. Thedithiocarbamate disulfide or thiolate anion and the heavy metal ion canbe administered in combination or separately. Preferably, they areadministered as a chelating complex. As is known in the art,dithiocarbamates are excellent chelating agents and can chelate heavymetal ions to form chelates. Preparation of chelates of dithiocarbamatesand heavy metal ions are known to the ordinary artisan. For example,chelates of diethyldithiocarbamate and copper, zinc, silver, or goldions can be conveniently synthesized by mixing, in suitable solvents,disulfiram or sodium diethyldithiocarbamate with, e.g., CuSO₄, ZnCl₂,C₃H₅AgO₃, or HAuCl₄.3H₂O to allow chelates to be formed. Otherdithiocarbamate-heavy metal ion chelates are disclosed in, e.g., D.Coucouvanis, “The chemistry of the dithioacid and 1,1-dithiolatecomplexes,” Prog. Inorganic Chem. 11:234-371 (1970); D. Coucouvanis,“The chemistry of the dithioacid and 1,1-dithiolate complexes,1968-1977,” Prog. Inorganic Chem. 26:302-469 (1978); R. P. Burns, etal., “1,1-ithiolato complexes of the transition metals,” Adv. InorganicChem. and Radiochem. 23:211-280(1980); L. I. Victoriano, et al., “Thereaction of copper (II) chloride and tetralkythiuram disulfides,” J.Coord. Chem. 35:27-34 (1995); L. I. Victoriano, et al., “Cuprousdithiocarbamates. Syntheses and reactivity,” J. Coord. Chem. 39:231-239(1996), which are incorporated herein by reference.

In accordance with another aspect of this invention, a method fortreating cancer in a patient is provided which includes administering tothe patient a therapeutically effect amount of a dithiocarbamate anioncompound and an intracellular heavy metal ion stimulant, which canenhance the intracellular level of the above described heavy metal ionsin the patient.

Intracellular heavy metal ion carriers are known. For example,ceruloplasmin can be administered to the patient to enhance theintracellular copper level. Other heavy metal ion carriers known in theart may also be administered in accordance with this aspect of theinvention. The heavy metal ion carriers and the dithocarbamate disulfideor thiolate anion can be administered together or separately, andpreferably in separate compositions.

Ceruloplasmin is a protein naturally produced by the human body and canbe purified from human serum. This 132-kD glycoprotein, which carries 7copper atoms complexed over three 43-45 kD domains, is an acute phasereactant and the major copper-carrying protein in human plasma. See,Halliwell, et al., Methods Enzymol. 186:1-85 (1990). When transportedinto cells, at least some of the bound cupric ions can be accessible forcomplexation with the dithiocarbamate disulfide or thiolate anionadministered to the patient. See, Percival, et al., Am. J. Physiol.258:3140-3146 (1990). Ceruloplasmin and dithiocarbamate disulfides orthiolate anions are typically administered in different compositions.Dithiocarbamate disulfides or thiolate anions can be administered atabout the same time, or at some time apart. For example, ceruloplasmincan be administered from about five minutes to about 12 hours before orafter dithiocarbamate disulfide or thiolate anions are administered tothe patient.

In another embodiment, instead of heavy metal ion carriers, a cytokineis administered to the patient in addition to a dithiocarbamatedisulfide or corresponding thiolate anion. Suitable cytokines include,e.g., interferon α, interferon β, interferon γ, and interleukin 6(IL-6). Such cytokines, when administered to a patient, are capable ofinducing an acute phase response in the body of the patient, thusstimulating elevations of serum ceruloplasmin in the patient.

The biochemical and physiological properties of such cytokines have beenstudied extensively in the art and are familiar to skilled artisans. Thecytokines can be purified from human or animal serum. They can also beobtained by genetic engineering techniques. In addition, commerciallyavailable samples of the above-identified cytokines may also be used inthis invention. Genetically or chemically modified cytokines can also beadministered. For example, it is known that certain peptide cytokineshave longer circulation time in animals when such cytokines areconjugated with a water soluble, non-immunogenic polymer such aspolyethylene glycol.

Typically, the cytokines are administered in a different compositionfrom the dithiocarbamate disulfide or corresponding thiolate anion. Thecytokines and dithocarbamate disulfide or thiolate anion can beadministered at about the same time, or at some time apart from eachother. For example the cytokines can be administered from about 5minutes to about 24 hours before or after the administration ofdithiocarbamate disulfide or thiolate anion.

In accordance with another aspect of this invention, the method of thisinvention can be used in combination with a conventional cancerchemotherapy, with the result that the treatment with dithiocarbamatedisulfides or thiolate anions, with or without heavy metals separatelyor as dithocarbamate-heavy metal chelates, will increase the sensitivityof the tumor to conventional cancer chemotherapy and result in greatereffectiveness of the conventional cancer chemotherapy drug. For example,the method of this invention can be complemented by a conventionalradiation therapy or chemotherapy. Thus, in one embodiment of thisinvention, the method of this invention comprises administering to apatient a dithiocarbamate disulfide compound or corresponding thiolateanion and heavy metals, and another anticancer agent. Treatment byceruloplasmin or a cytokine, and a dithiocarbamate disulfide or thiolateanion can also be conducted along with the treatment with anotheranticancer agent to increase the effectiveness of the anticancer agent.

Any anticancer agents known in the art can be used in this invention solong as it is pharmaceutically compatible with the dithiocarbamatedisulfide or thiolate anion compound, heavy metal ion, ceruloplasmin,and/or cytokines used. By “pharmaceutically compatible” it is intendedthat the other anticancer agent will not interact or react with theabove composition, directly or indirectly, in such a way as to adverselyaffect the effect of the treatment of cancer, or to cause anysignificant adverse side reaction in the patient.

Exemplary anticancer agents known in the art include busulphan,chlorambucil, hydroxyurea, ifosfamide, mitomycin, mitotane,chlorambucil, mechlorethamine, carmustine, lomustine, cisplatin,carmustine, herceptin, carboplatin, cyclophosphamide, nitrosoureas,fotemustine, vindescine, etoposide, daunorubicin, adriamycin,paclitaxel, docetaxel, streptozocin, dactinomycin, doxorubicin,idarubicin, plicamycin, pentostatin, mitotoxantrone, valrubicin,cytarabine, fludarabine, floxuridine, clardribine, methotrexate,mercaptopurine, thioguanine, capecitabine, irinotecan, dacarbazine,asparaginase, gemcitabine, altretamine, topotecan, procarbazine,vinorelbine, pegaspargase, vincristine, rituxan, vinblastine, tretinoin,teniposide, fluorouracil, melphalan, bleomycin, salicylates, aspirin,piroxicam, ibuprofen, indomethacin, naprosyn, diclofenac, tolmetin,ketoprofen, nambuetone, oxaprozin, doxirubicin, nonselectivecycclooxygenase inhibitors such as nonsteroidal anti-inflammatory agents(NSAIDS), and selective cyclooxygenase-2 (COX-2) inhibitors.

The anticancer agent used can be administered simultaneously in the samepharmaceutical preparation with the dithiocarbamate disulfide orthiolate anion compound, heavy metal compounds or dithiocarbamate-heavymetal chelates, ceruloplasmin, and/or cytokines as described above. Theanticancer agent can also be administered at about the same time but bya separate administration. Alternatively, the anticancer agent can beadministered at a different time from the administration of thedithiocarbamate disulfide or thiolate anion compound, heavy metalcompounds or dithiocarbamate-heavy metal chelates, ceruloplasmin, and/orcytokines. Some minor degree of experimentation may be required todetermine the best manner of administration, this being well within thecapability of one-skilled in the art once apprised of the presentdisclosure.

The methods of this invention a particularly useful in treating humans.Also, the methods of this invention are suitable for treating cancers inanimals, especially mammals such as canine, bovine, porcine, and otheranimals. The methods are useful for treating various types of cancer,including but not limited to melanoma, non-small cell lung cancer, smallcell lung cancer, renal cancer, colorectal cancer, breast cancer,pancreatic cancer, gastric cancer, bladder cancer, ovarian cancer,uterine cancer, lymphoma, and prostate cancer. In particular, thepresent invention will be especially effective in treating melanoma,lung cancer, breast cancer, and prostate carcinoma.

The active compounds of this invention are typically administered in apharmaceutically acceptable carrier through any appropriate routes suchas parenteral, intravenous, oral, intradermal, subcutaneous, or topicaladministration. The active compounds of this invention are administeredat a therapeutically effective amount to achieve the desired therapeuticeffect without causing any serious adverse effects in the patienttreated.

The dithiocarbamate disulfide compound disulfiram and itsdiethyldithiocarbamate thiolate anion are effective when administered atamounts within the conventional clinical ranges determined in the art.Disulfiram approved by the U.S. Food and Drug administration (Antabuse®)can be purchased in 250 and 500 mg tablets for oral administration fromWyeth-Ayerst Laboratories in Philadelphia, Pa. 19101. Typically, it iseffective at an amount of from about 125 to about 1000 mg per day,preferably from 250 to about 500 mg per day for disulfiram and 100 to500 mg per day or 5 mg/kg in travenously or 10 mg/kg orally once a weekfor diethyldithiocarbamate. However, the dosage can vary with the bodyweight of the patient treated. The active ingredient may be administeredat once, or may be divided into a number of smaller doses to beadministered at predetermined intervals of time. The suitable dosageunit for each administration of disulfiram is, e.g., from about 50 toabout 1000 mg/day, preferably from about 1250 to about 500 mg/day. Thedesirable peak concentration of disulfiram generally is about 0.05 toabout 10 μM, preferably about 0.5 to about 5 μM, in order to achieve adetectable therapeutic effect. Similar concentration ranges aredesirable for dithiocarbamate thiolate anions and fordithocarbamate-heavy metal complexes.

Disulfiram implanted subcutaneously for sustained release has also beenshown to effective at an amount of 800 to 1600 mg to achieve a suitableplasma concentration. This can be accomplished by using aseptictechniques to surgically implant disulfiram into the subcutaneous spaceof the anterior abdominal wall. See, e.g., Wilson, et al., J. Clin.Psych. 45:242-247 (1984). In addition, sustained release dosageformulations, such as an 80% poly(glycolic-co-L-lactic acid) and 20%disulfiram, may be used. The therapeutically effective amount for otherdithiocarbamate disulfide compounds may also be estimated or calculatedbased on the above dosage ranges of disulfiram and the molecular weightsof disulfiram and the other dithiocarbamate disulfide compound, or byother methods known in the art.

The diethyldithiocarbamate thiolate anion has not been previouslyadvocated as a cancer chemotherapeutic agent itself, nor has it beensuggested as a treatment to increase the sensitivity of tumors to cancerchemotherapy drugs. For the treatment of HIV infection, humans have beentreated with doses of 5 mg/kg intravenous or 10 mg/kg orally, once aweek. Minimal side effects on this dosage regimen include a metallictaste in the mouth, flatulence and intolerance to alcoholic beverages.An enteric-coated oral dosage form of diethyldithiocarbamate thiolateanions to liberate active drug only in the alkaline environment of theintestine is preferred because of the potential for liberation of carbondisulfide upon exposure of diethyldithiocarbamate to hydrochloric acidin the stomach. An oral enteric-coated form of sodiumdiethyldithiocarbamate is available in 125 mg tablets as Imuthiol®through Institute Merieux, Lyon, France.

Heavy metal ions can be administered separately as an aqueous solutionin a pharmaceutically suitable salt form. The salt form is ideally achelate with an organic anion such as acetate, lactonate, glycinate,citrate, propionate or gluconate in order to enhance absorption.However, the heavy metals are preferably administered in a chelate formin which the ions are complexed with the dithiocarbamate as a1,1-dithiolate complex. Thus, the amount of heavy metal ions to be usedadvantageously is proportional to the amount of dithiocarbamatedisulfide compound to be administered based on the molar ratio between aheavy metal ion and the dithiocarbamate in the chelate. Methods forpreparing such chelates or complexes are known and the preferred methodsare disclosed above and in the examples below.

The therapeutically effective amount of IL-6 can be from about 1 toabout 100 μg/kg per day, preferably from about 5 to about 50 μg/kg perday. Interferon α can be administered at from about 0.1×10⁶ to about10×10⁶ international units per day, preferably from about 3 to about8×10⁶ international units per day, and the administration frequency canbe from about three times per week to about once per day. Suitabledosage for interferon β can range from about 1 to about 200 μg per day,preferably from about 10 to about 100 μg per day administered once perweek up to once per day. Interferon γ can be administered at a dosage offrom about 1 to about 1000 μg per day, preferably from about 50 to about250 μg per day. Ceruloplasmin may be administered at an amount of fromabout 1 to about 100 mg per day, preferably from about 50 to about 30 mgper day.

It should be understood that the dosage ranges set forth above areexemplary only and are not intended to limit the scope of thisinvention. The therapeutically effective amount for each active compoundcan vary with factors including but not limited to the activity of thecompound used, stability of the active compound in the patient's body,the severity of the conditions to be alleviated, the total weight of thepatient treated, the route of administration, the ease of absorption,distribution, and excretion of the active compound by the body, the ageand sensitivity of the patient to be treated, and the like, as will beapparent to a skilled artisan. The amount of administration can also beadjusted as the various factors change over time.

Advantageously, the active compounds are delivered to the patientparenterally, i.e., intravenously or intramuscularly. For parenteraladministration, the active compounds can be formulated into solutions orsuspensions, or in lyophilized forms for conversion into solutions orsuspensions before use. Sterile water, physiological saline, e.g.,phosphate buffered saline (PBS) can be used conveniently as thepharmaceutically acceptable carriers or diluents. Conventional solvents,surfactants, stabilizers, pH balancing buffers, anti-bacteria agents,and antioxidants can all be used in the parenteral formulations,including but not limited to acetates, citrates or phosphate buffers,sodium chloride, dextrose, fixed oils, glycerine, polyethylene glycol,propylene glycol, benzyl alcohol, methyl parabens, ascorbic, acid,sodium bisulfite, and the like. For parenteral administration, theactive compounds, particularly dithiocarbamate-metal chelates, can beformulated contained in liposomes so as to enhance absorption anddecrease potential toxicity. The parenteral formulation can be stored inany conventional containers such as vials, ampoules, and syringes.

The active compounds can also be delivered orally in enclosed gelatincapsules or compressed tablets. Capsules and tablets can be prepared inany conventional techniques. For example, the active compounds can beincorporated into a formulation which includes pharmaceuticallyacceptable carriers such as excipients (e.g., starch, lactose), binders(e.g., gelatin, cellulose, gum), disintegrating agents (e.g., alginate,Primogel, and corn starch), lubricants (e.g., magnesium stearate,silicon dioxide), and sweetening or flavoring agents (e.g., glucose,sucrose, saccharin, methyl salicylate, and peppermint). Various coatingscan also be prepared for the capsules and tablets to modify the flavors,tastes, colors, and shapes of the capsules and tablets. In addition,liquid carriers such as fatty oil can also be included in capsules. Foradministration of dithiocarbamate thiolate anions anddithiocarbamate-metal complexes, it is desirable to administer thecompounds as enteric-coated capsules that are impervious to stomach acidbut dissolve in the alkaline environment of the small intestine, inorder to prevent release of carbon disulfide from dithiocarbamates inthe acid environment of the stomach, and to preserve the integrity ofthe dithiocarbamate-metal chelate.

Other forms of oral formulations such as chewing gum, suspension, syrup,wafer, elixir, and the like can also be prepared containing the activecompounds used in this invention. Various modifying agents for flavors,tastes, colors, and shapes of the special forms can also be included. Inaddition, for convenient administration by enteral feeding tube inpatients unable to swallow, the active compounds can be dissolved in anacceptable lipophilic vegetable oil vehicle, such as olive oil, cornoil, and safflower oil.

The active compounds can also be administered topically through rectal,vaginal, nasal or mucosal applications. Topical formulations aregenerally known in the art including creams, gels, ointments, lotions,powders, pastes, suspensions, sprays, and aerosols. Typically, topicalformulations include one or more thickening agents, humectants, an/oremollients including but not limited to xanthan gum, petrolatum,beeswax, or polyethylene glycol, sorbitol, mineral oil, lanolin,squalene, and the like. A special form of topical administration isdelivery by a transdermal patch. Methods for preparing transdermalpatches are disclosed, e.g., in Brown, et al., Annual Review ofMedicine. 39:221-229 (1988), which is incorporated herein by reference.

The active compounds can also be delivered by subcutaneous implantationfor sustained release. This may be accomplished by using aseptictechniques to surgically implant the active compounds in any suitableformulation into the subcutaneous space of the anterior abdominal wall.See, e.g., Wilson, et al., J. Clin. Psych. 45:242-247 (1984). Sustainedrelease can be achieved by incorporating the active ingredients into aspecial carrier such as a hydrogel. Typically, a hydrogel is a networkof high molecular weight biocompatible polymers, which can swell inwater to form a gel like material. Hydrogels are generally known in theart. For example, hydrogels made of polyethylene glycols, or collage, orpoly(glycolic-co-L-lactic acid) are suitable for this invention. See,e.g., Phillips, et. al., J. Pharmceut. Sci. 73:1718-1720 (1984).

The active compounds can also be conjugated, i.e., covalently linked, toa water soluble non-immunogenic high molecular weight polymer to form apolymer conjugate. Advantageously, such polymers, e.g., polyethyleneglycol, can impart solubility, stability, and reduced immunogenicity tothe active compounds. As a result, the active compound in the conjugatewhen administered to a patient, can have a longer half-life in the body,and exhibit better efficacy. PEGylated proteins are currently being usedin protein replacement therapies and for other therapeutic uses. Forexample, PEGylated adenosine deaminase. (ADAGEN®) is being used to treatsevere combined immunodeficiency disease (SCIDS). PEGylatedL-asparaginase (ONCAPSPAR®) is being used to treat acute lymphoblasticleukemia (ALL).

Alternatively, other forms of controlled release or protection includingmicrocapsules and nanocapsules generally known in the art, and hydrogelsdescribed above can all be utilized in oral, parenteral, topical, andsubcutaneous administration of the active compounds.

As discussed above, another preferable delivery form is using liposomesas a carrier. Liposomes are micelles formed from various lipids such ascholesterol, phospholipids, fatty acids and derivatives thereof. Activecompounds can be enclosed within such micelles. Methods for preparingliposomal suspensions containing active ingredients therein aregenerally known in the art and are disclosed in, e.g., U.S. Pat. No.4,522,811, which is incorporated herein by reference. Several anticancerdrugs delivered in the form of liposomes are known in the art and arecommercially available from Liposome, Inc., of Princeton, N.J. It hasbeen shown that liposomal delivery can reduce the toxicity of the activecompounds, and increase their stability.

The active compounds can also be administered in combination with otheractive agents that treat or prevent another disease or symptom in thepatient treated. However, it is to be understood that such other activeagents should not interfere with or adversely affect the effects of theactive compounds of this invention on the cancer being treated. Suchother active agents include but are not limited to antiviral agents,antibiotics, antifungal agents, anti-inflammation agents, antithromboticagents, cardiovascular drugs, cholesterol lowering agents, hypertensiondrugs, and the like.

It is to be understood that individuals placed on dithiocarbamatedisulfide or thiolate anion therapy for their cancer in any form must bewarned against exposure to alcohol in any form, to avoid theprecipitation of nausea and vomiting from buildup of acetaldehyde in thebloodstream. Subjects therefore must not only refrain from ingestingalcohol containing beverages, but should also not ingest over thecounter formulations such as cough syrups containing alcohol or even userubbing alcohol topically.

Experimental Procedures

Materials.

Human malignant cell lines were obtained from American Type TissueCulture Collection (Rockville, Md.). RPMI medium 1640, Leibovitz's L-15medium, N-2-hydroyethylpiperazine-N′-2-ethanesulfonic acid (HEPES),antibiotic-antimycotic (10,000 U penicillin, 10,000 μg streptomycin, and25 μg amphotericin B/ml), fetal bovine serum (FBS) andtrypsin-ethylenediaminetetraacetic acid (EDTA) solution were purchasedfrom the GIBCO-BRL division of Life Technologies (Grand Island, N.Y.).Rabbit polyclonal antibodies against human Bcl-2, p53, p21^(WAF1Cip1)cyclins A and B1, CREB1, ATF1, ATF2, c-Jun and Jun B were from SantaCruz Biotechnology (Santa Cruz, Calif.). Rabbit polyclonal antibodyagainst c-Fos and A431 cell lysate standard were from Calbiochem (SanDiego, Calif.). Peroxidase-labeled donkey polyclonal anti-rabbit IgG wasfrom Amersham Life Sciences (Buckinghamshire, England), andperoxidase-labeled anti-goat IgG was from Santa Cruz Biotechnology.Electrophoretic mobility shift assay (EMSA) supplies, including DNAprobes, were purchased from ProMega (Madison, Wis.). Protease inhibitorswere from Boehringer Mannheim (Indianapolis, Ind.). The diacetate of2′,7′-dichlorofluorescin (DCF-DA) was purchased from Molecular Probes(Eugene, Oreg.). Pyrrolidinedithiocarbamate (PDTC),diethyldithiocarbamate, tetraethylthiuram disulfide (di sulfiram),bathocuproinedisulfonic acid (BCPS), metal salts, nonenzymatic CellDissociation Solution®, Nω-nitro-L-arginine, indomethacin, bovine serumalbumin (BSA) and all other materials were purchased from Sigma ChemicalCo. (St. Louis, Mo.), unless specified.

Culture of Malignant Cell Lines.

Human malignant cell lines were obtained from American Type TissueCulture Collection (Rockville, Md.). Melanoma cells lines CRL 1585 and1619 were cultured in RPMI1640 (GIBCO-BRL, Life Technologies, GrandIsland, N.Y.) with 10% FBS and passed with nonenzymatic CellDissociation Solution® (Sigma). The prostate adenocarcinoma cell lineCRL 1435 (PC-3) was also cultured in RPMI 1640 with 10% FBS but passedwith 0.05% trypsin and 0.53 mM EDTA. The squamous lung carcinomaNCI-H520 and the adenosquamous lung carcinoma NCI-H596 cell lines weregrown in RPMI1640 supplemented with 10% FBS, 10 mM HEPES and 1.0 mMsodium pyruvate and passed with trypsin/EDTA. The small cell lungcarcinoma NCI-H82 was cultured as a suspension in RPMI 1640 with 10%FBS. All of the above were grown in a 37° C. humidified environmentcontaining 5% CO₂/air. The breast carcinoma cell line MDA-MB-453 wasgrown in a 37° C. humidified environment with free gas exchange withatmospheric air using Leibovitz's L-15 medium with 2 mM L-glutamine and10% FBS and was passed with trypsin/EDTA.

Cell Culture Treatments.

Because the disulfide form of dithiocarbamates does not have a freethiol to act as an antioxidant, most of the experiments were performedwith the tetraethylthiuram disulfide disulfiram. To study the effect ofdisulfiram on activation of select genes important for cellularproliferation, malignant melanoma cells were grown to confluence on100×15 mm plastic Petri dishes and treated with 5 μM disulfiram or 5 μMdisulfiram plus 1.6 μM CuSO₄. This dose was chosen to approximate thesteady state plasma and tissue concentrations of drug in human subjectson chronic therapy with this agent. Disulfiram was solubilized indimethylsulfoxide (DMSO) so that the final concentration of DMSO wasless than 0.3-0.5%. Equal volumes of DMSO were added to controlexperiments. Nuclear protein was harvested and electrophoretic mobilitygel shift assays were performed using DNA consensus binding sequence forthe cyclic-AMP responsive element (CRE) as outlined below. To determinewhether disulfiram and metals might directly influence transcriptionfactor binding, in some experiments, 5 μM disulfiram and/or CuSO₄ 1.6 μMCuSO₄ (final concentrations) were added to the binding reaction ofnuclear protein obtained from control cells stimulated with 10% FBSalone in the absence of drugs or metals. In vitro addition of disulfiramand CuSO₄ to the binding reaction was performed using either 2.5 mMdithiothreitol (DTT) or 3.0 mM GSH as a reducing agent in the bindingbuffer.

The effect of disulfiram (0.15 to 5.0 μM), diethyldithiocarbamate (DDC,1.0 μM) or PDTC (0.625 to 5.0 μM) on proliferation of malignant celllines was studied in cultures stimulated with 10% FBS. Cell numbers werequantitated 24-72 hours later. In some experiments disulfiram or PDTCwere added immediately after cells were plated. In other experiments,cells were plated and allowed to grow for 24-72 hours before fresh mediawith disulfiram or PDTC was added, and cell numbers were assayed 24-72hours later. Synergy was studied between disulfiram andN,N′-bis(2-chloroethyl-N-nitrosourea (carmustine or BCNU, 1.0 to 1,000μM) or cisplatin (0.1 to 100 μg/ml) added to medium. The effect ofmetals on disulfiram was studied with 0.2 to 10 μM copper (provided asCuSO₄), zinc (as ZnCl₂), silver (as silver lactate) or gold (asHAuCl₄₋₃H₂O) ions added to growth medium. No pH changes occurred withaddition of metal salts to culture medium. To provide a biologicallyrelevant source of copper, in some experiments medium was supplementedwith human ceruloplasmin at doses replicating low and high normal adultserum concentrations (250 and 500 μg/ml).

Potential redox effects of disulfiram were studied in three sets ofexperiments. The importance of cellular glutathione (GSH) in mediatingor modulating thiocarbamate toxicity was studied by measuring levels ofintracellular GSH after treatment with disulfiram. Disulfiram (5 μM),with or without 1.6 μM CuSO₄, was added to cells grown to confluence on100×15 mm plastic dishes, and cells were harvested 24 hour later formeasurement of GSH as outlined below. Also, to assess whether anonspecific antioxidant effect of disulfiram or PDTC might account forcellular growth inhibition, we studied the effect of the potentlipophilic antioxidant probucol (1.0 to 1,000 μM) on proliferation ofmalignant cell lines. Finally, the generation of intracellular oxidantsin response to disulfiram (0.625 to 5 μM), copper (0.2 to 1.6 μM CuSO₄)or 1.25 μM disulfiram plus various concentration of copper was measureddirectly.

To explore the role of cyclooxygenase inhibition on tumor cell growth,cells were cultured with or without disulfiram in the presence orabsence of the cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2)inhibitors indomethacin (5 μg/ml) or sodium salicylate (1 mM). To probewhether disulfiram might be inducing growth retardation by interruptionor stimulation of NO production, proliferation was studied with andwithout disulfiram in the presence and absence of the nitric oxidesynthese inhibitor Nω-nitro-L-arginine added to growth medium (100 μM).

Finally, a number of dithiocarbamate effects on cells have beenattributed to increasing the intracellular levels of copper ions. Tofurther probe the role of copper in mediating cytotoxicity fromdisulfiram, cells were cultured with or without addition of theimpermeate CuCu²⁺ chelator bathocuprioinedisulfonic acid (BCPS, 100 μM)added to medium to sequester CuCu²⁺ in the extracellular compartment.Cells were also treated 12 hours with various concentration ofdisulfiram (0.625 to 5.0 μM) and intracellular copper levels weremeasured as outlined below.

Electrophoretic Mobility Shift Assays (EMSAs).

Nuclear protein was isolated and DNA binding reactions were performed aspreviously described in detail (See, e.g., R. Dashtaki, et al.,“Dehydroepiandrosterone and analogs inhiibit DNA binding of AP-1 andairway smooth muscle proliferation,” J. Pharmacol. Exper. Ther.285:876-219 (1998); T. Kennedy, et al., “Copper-dependent inflammationand nuclear factor-κB activation by particulate air pollution,” Am. J.Respir. Cell Mol. Biol. 19:366-378 (1998)). Monolayers were washed twicein cold DPBS and equilibrated 10 minutes on ice with 0.7 ml coldcytoplasmic extraction buffer, CEB (10 mM Tris, pH 7.9,60 mM KCl, 1 mMEDTA, 1 mM DTT) with protease inhibitors, PI (1 mM Pefabloc, 50 μg/mlantipain, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 40 μg/ml bestatin, 3μg/ml E-64 and 100 μg/ml chymostatin). The detergent Nonidet P-40(NP-40) was added to a final concentration of 0.1% and cells weredislodged with a cell scraper. Nuclei were pelleted by centrifugationand washed with CEB/PI. Nuclei were then incubated for 20 minutes on icein nuclear extraction buffer, NEB (20 mM Tris, pH 8.0, 400 mM NaCl, 1.5mM MgCl₂, 1.5 mM EDTA, 1 mM DTT and 25% glycerol) with PI, spun brieflyto clear debris and stored at −80° C. until performance ofelectrophoretic mobility shift assays.

EMSAs were performed using consensus oligonucleotides(5′-AGAGATTGCCTGACGTCAGAGAGCTAG-3′and 3′-TCTCTAACGGACTGCAGTCTCTCGATC-5′)for the cyclic-AMP responsive element CRE (ProMega, Madison, Wis.),end-labeled by phosphorylation with [γ³²P]-ATP and T4 polynucleotidekinase. DNA-protein binding reactions were performed with 2 μg ofnuclear protein (as determined by the Pierce method) and 30-80,000 cpmof ³²P-end-labelled double-stranded DNA probe in 10 mM Tris-HCl, pH 7.5,50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT (except where indicated), 1 mMMgCl₂, 50 μg/ml poly dI-dC, and 4% glycerol. All components of thebinding reaction with the exception of labeled probe were combined andincubated at room temperature for 10 minutes before addition of labeledprobe and incubation for an additional 20 minutes.

Competition experiments were performed with 10× unlabeled wild-typeoligonucleotide sequences for CRE or NF-κB (p50,5′-AGTTGAGGGGACTTTCCCAGGC-3′and 3′-TCAACTCCCCTGAAAGGGTCCG-5′), addedbefore labeled probe. Supershift experiments were performed byincubating the binding reaction with 1 μg of supershifting antibodyprior to electrophoresis. Samples were electrophoresed on a 5%nondenaturing polyacrylamide gel in Tris-glycine-EDTA (TGE, 120 mMglycine and 1 mM EDTA in 25 mM Tris, pH 8.5) buffer. Gels were dried andanalyzed by autoradiography at −80° C. using an image intensifierscreen. Densitometry of bands was performed using Kodak Digital Science1D image analysis software (Eastman Kodak, Rochester, N.Y.).

Measurement of Proliferation in Cell Cultures

Proliferation of cultured cells was quantitated using a previouslyreported colorimetric method based upon metabolic reduction of thesoluble yellow tetrazolium dye 3-[4,5-dimnethylthiazol]-2yl-2,5-diphenyltetrazolium bromide (MTT) to its insoluble purple formazan by the actionof mitochondrial succinyl dehydrogenase (See, e.g., S. J. Hirst, et al.,“Quantifying proliferation of cultured human and rabbit airway smoothmuscle in response to serum and platelet derived growth factor,” Am. J.Respir. Cell Mol. Biol. 7:574-581 (1992); R. Dashtaki, et al. R., supra;S. S. Brar, et al., “Requirement for reactive oxygen species inserum-induced and platelet-derived growth factor-induced growth ofairway smooth muscle,” J. Biol. Chem. 274:20017-20026(1999)). This assayempirically distinguishes between dead and living cells. Forproliferation studies, cells were seeded into 24-well uncoated plasticplates (Costar) at 50,000 cells per well and cultured with respectivemedia and mitogens. After 24-96 hours, medium was removed, cells werewashed twice with 1 ml of sterile Dulbecco's modified phosphate bufferedsaline without CaCu²⁺ or MgCu²⁺ (DPBS), the medium was replaced with 1ml/well fresh medium containing 100 μg/ml MTT, and plates were incubatedan additional hour. MTT-containing medium was removed, 0.5 mldimethylsulfoxide (DMSO) was added to each well, and the absorbance ofthe solubilized purple formazan dye was measured at 540 nm. A total of4-6 wells were studied at each treatment condition. Preliminary studieswere performed with 50-200 μg/ml MTT incubated for 15 minutes to 3 hoursto determine the optimum concentration and incubation time at which therate of conversion was linear and proportional to the number of cellspresent. The absorbence of the MTT formazan reduction product (A₅₄₀)correlated with cell numbers counted by hemocytometer with an R²=0.99.In some experiments, the MTT assay and responses to FBS and inhibitorswere also confirmed by performing cell counts on 10 random fields/wellof Giemsa-modified Wright's stained monolayers viewed at 40 power usinga 0.01-cm² ocular grid.

Measurement of Cytotoxicity and Apoptosis

To assess for cytotoxicity, cells were plated at a density of 50,000 perwell on 24 well plates and grown for 24 hours. Disulfiram was thenadded. After an additional 36 hours, medium was removed and replacedwith DPBS containing 0.1% trypan blue. Cell death was assessed bycounting the average number of trypan blue positive cells per 10× fieldin 5 random fields for 4 separate wells.

To determine whether disulfiram induced apoptosis, cells grown toconfluence on 35 mm Petri dishes or on glass slides were treated withdisulfiram or DMSO as vehicle. Apoptosis was studied by terminaldeoxynucleotidyl transferase (TdT) dependent 3′-OH fluoresceinend-labeling of DNA fragments, using a Fluorescein-FragEL™ DNAfragmentation detection kit (Oncogene Research Products, Cambridge,Mass.). Apoptosis was also studied by visually assessing endonucleasedependent DNA fragmentation on ethidium bromide-stained agarose gels.

DNA Cell Cycle Measurements

To study the effect of disulfiram on the DNA cell cycle, cells weregrown to confluence in 25 cm² plastic flasks and treated for with 10%FBS plus DMSO vehicle, FBS and DMSO vehicle plus 250 μg/ml ceruloplasminas a source of copper, FBS plus 5 μM disulfiram or FBS plus 5 μMdisulfiram and 250 μg/ml ceruloplasmin. After 24 hours cells weretrypsinized, washed twice in cold DPBS with 1 mM EDTA and 1% BSA, fixed30 minute in ice-cold 70% ethanol, and stained by incubation for 30minutes at 37° C. in a 10 μg/ml solution of propidium iodide in DPBS and1 mg/ml RNase A. DNA cell cycle measurements were made using aFACStar^(PLUS) Flow Cytometer (Becton-Dickenson, San Jose, Calif.).

Immunossay for Proteins

Cells were lysed and proteins were isolated and quantitated byimmunoassay as previously detailed using 2 μg/ml of primary rabbitpolyclonal antibodies against human bcl-2, p53, p21^(WAF1/Cip1), cyclinA and cyclin B1, and peroxidase-labeled donkey polyclonal, anti-rabbitIgG. Cells were placed on ice, washed twice with cold DPBS, scraped into0.5 ml boiling buffer (10% [vol/vol] glycerol and 2% [wt/vol] sodiumdodecyl sulfate [SDS] in 83 mM Tris, pH 6.8) and sheared by fourpassages through a pipette. Aliquots were removed for proteindetermination, using the BCA protein assay (Pierce). After 10%β-mercaptoethanol and 0.05% bromophenol blue were added, lysates wereboiled for 5 min and stored at −80° C. until immunoblotting wasperformed. Proteins in defrosted samples were separated bySDS-polyacrylamide gel electrophoresis on 12% polyacrylainide gels (15μg protein/lane) and electrotransferred to 0.45 μm Hybond ECLnitrocellulose membranes (Amersham Life Sciences) using the wettransblot method in transfer buffer (0.025 M Tris, 0.192 M glycine, 2.6mM SDS, and 20%[vol/vol] methanol; pH 8.8) at 100 volts for 1 hour.Blots were blocked overnight at 4° C. with blocking buffer (PBS with0.1% Tween 20) containing 5% fat-free milk powder (Carnation, Glendale,Calif.). After rinsing 5 times for 5 minutes each in PBS containing 0.1%Tween 20, blots were incubated for 1 hour at room temperature with 2.0μg/ml of primary antibody. After rinsing again as above, blots wereincubated for 1 hr at room temperature with horseradishperoxidase(HRP)-conjugated secondary antibody diluted 1:5,000 inblocking buffer. Immunoblots were rinsed again as above and detected viaan enhanced chemiluminescence method (ECL Western blotting detectionsystem, Amersham Life Science, Buckinghamshire, England).Autoradiographic film (X-OMAT AR, Eastman Kodak, Rochester, N.Y.) wasexposed to immunoblots for 10, 30, or 60 seconds to obtain satisfactoryimages.

Measurement of Intracellular Copper

Cells were cultured in 12-well plastic tissue culture plates at aninitial plating density of 50,000 cells/well, grown to confluence andtreated with disulfiram or vehicle DMSO as outlined above. Media wasremoved and cells were washed twice with DPBS. Cells were then scrapedinto 1.0 ml of 3N HCl/10.0% trichloroacetic acid and hydrolyzed at 70°C. for 16 hours. The hydrolysate was centrifuged at 600 gm for 10minutes to remove debris and copper was measured in the supernatantusing inductively coupled lasma emission spectroscopy (Model P30, PerkinElmer, Norwalk, Conn.) at wavelengths of 325.754 and 224.700 nm. Tominimize metal contamination, plasticware rather than glassware was usedin these experiments, and double-distilled, deionized water was used forall aqueous media. Results are reported as ng copper/ml of hydrolysate.

Measurement of Intracellular Generation of Reactive Oxygen Species

Generation of reactive oxygen species in response to disulfiram with orwithout CuSO₄ was studied using 2′,7′-dichlorofluorescin diacetate(DCF-DA, Molecular Probes, Eugene, Org.) and a modification of methodspreviously reported (See, J. A. Royall, et al., “Evaluation of2′,7′-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probesfor intracellular H₂O₂ in cultured endothelial cells,” Archiv. Biochem.Biophys. 302:348-355 (1993)). This method is based upon oxidation ofdichlorofluorescin to 2′,7′-dichlorofluorescein by H₂O₂ in the presenceof cellular peroxidases. Cells were plated in 24 well plastic plates at50,000 cells per well and grown to confluence. Media was aspirated fromwells and replaced with 100 μl medium containing 10 μM DCF-DA, andplates were incubated at 37° C. for 30 minutes. The DCF-DA containingmedia was aspirated, cells were washed twice with media alone and 100 μlfresh media was added to wells. With the plate on the fluorescencemicro-plate reader (HTS 7000) cells were stimulated with 25 μl of mediacontaining 5 × concentrations of disulfiram and/or CuSO₄ to providefinal concentrations of 0-5.0 μM disulfiram and/or 0-1.6 μM CuSO₄,respectively. The relative concentration of dichlorofluroescein wasmeasured immediately by monitoring fluorescence at 37° C. using anexcitation wavelength of 485 nm and emission wavelength of 535 nm.

Measurement of Intracellular Glutathione

Disulfiram (5 μM), with or without 1.6 μM CuSO₄, was added to cellsgrown to confluence on 100×15 mm plastic dishes, and cells wereharvested 24 hours later for measurement of GSH using the5,5′-dithiobis(2-nitrobenzoic acid)-glutathione reductase recyclingassay (See, M. E. Anderson, “Determination of glutathione andglutathione disulfide in biological samples,” Methods Enzymol.113:548-555 (1985)).

Synthesis of Disulfiram-Metal Chelates

Chelates of disulfiram and a number of metals were synthesized byvigorous mixing of 150 mg of disulfiram in chloroform (7.5 mg/ml) with30 ml of a 5× molar excess of CuSO₄, ZnCl₂, C₃H₅AgO₃ (silver lactate) orHAuCl₄.3H₂O in double glass distilled deionized water. The mixture wascentrifuged at 1,000 μm for 10 minutes and the upper aqueous phase wasdiscarded. As the lower chloroform phase was evaporated, the resultingdisulfiram-metal chelates precipitated.

In another synthesis, 150 mg of sodium diethyldithiocarbamate wasdissolved in 10 ml of deionized water. To this was added 250 mg ofHAuCl₄.3H₂O. The resulting precipitate was collected by centrifugationand redisolved in chloroform. As the chloroform phase was evaporated theresulting dithiocarbamate-gold chelates were precipitated as crystals.

These were analyzed to determine their molecular weight, melting point,solubility, elemental composition and crystallographic structure.

Statistical Analysis

Data are expressed as mean values±standard error. The minimum number ofreplicates for all measurements was four, unless indicated. Differencesbetween multiple groups were compared using one-way analysis ofvariance. The post-hoc test used was the Newman-Keuls multiplecomparison test. Two-tailed tests of significance were employed.Significance was assumed at p<0.05.

EXAMPLE 1

This example shows dithiocarbamate disulfides inhibit DNA binding to thecyclic AMP response element.

M1619 melanoma cells were grown to 60% confluence on 100×15 mm plasticPetri dishes, nuclear protein was harvested and electrophoretic mobilitygel shift assays (EMSAs) were performed using. The results are shown inFIGS. 1A-1C. Treatment of cells for 6, 12 or 24 hour with thecombination of 5 μM disulfiram and 1.6 μM cupric sulfate substantiallyinterrupts transcription factor binding to CRE. EMSAs for 2, 6, 12 or 24hours of treatment: FBS alone, lanes 1, 5, 9, and 13; FBS+DMSO vehicle,lanes 2, 6, 10, 14; FBS+disulfiram, lanes 3, 7, 11, 15;FBS+disulfiram+CuSO₄, lanes 4, 8, 12, 16.

CRE complexes (I and II) are labeled. Nuclear protein from proliferatingM1619 malignant melanoma cells showed two strong constitutive bands (Iand II) of DNA binding activity in electrophoretic mobility shift assayswith the cyclic AMP response element (CRE) consensus sequence (FIG. 1A,lane 1). Both bands were eliminated by addition of 10× unlabeled CREconsensus oligonucleotide to the binding reaction (lane 8). Supershiftexperiments demonstrated that the top band II contains the CRE bindingprotein activating transcription factor-2 (ATF-2, lane 5), while thelower complex I contains CREB-1 (lane 2), with ATF-1 (lane 4) as a minorcomponent. Competition experiments shown in lanes 6-8 demonstratespecificity of the DNA binding reaction: lane 6, FBS :(fetal bovineserum) alone; lane 7, FBS with 10× unlabeled CRE probe added to bindingreaction; lane 8, FBS with 10× unlabeled NF-κB probe added to bindingreaction.

As shown in FIG. 1B, disulfiram alone slightly reduced DNA-binding toCRE, but when combined with treatment of cells with the transition metalcopper, disulfiram eliminated transcription factor binding to CRE after6 hours of treatment.

The upper ATF-2 containing complex proved more sensitive to inhibition.This is demonstrated in FIG. 1C, which shows densitometry resultsperformed on the ATF-2 containing upper complex II experiments isdisplayed as mean sum intensity of bands. The EMSAs in replicateexperiments (n=4) in which near confluent cells were treated for 8 hourswith DMSO vehicle, disulfiram, copper or the combination of disulfiramplus copper. The combination of disulfiram plus copper reduced DNAbinding of the upper complex II by half suggests that ATF-2 is extremelysensitive to inhibition by interactions between thiuramdisulfides andsome metals. At the concentrations employed above, disulfiram pluscopper also inhibited DNA binding of NF-κB after treatment for 12 hoursand DNA binding of AP-1 after 24 hours (data not shown), but effectswere not as dramatic those on binding to CRE.

To determine if inhibition of transcription factor binding to CRE couldbe attributed to direct transcription factor modification by disulfiramand copper, we studied the effect of adding each agent directly to thebinding reaction performed with nuclear protein from untreated M1619cells. The results are shown in FIG. 2. Therein electrophoretic mobilityshift assays (EMSAs) were performed showing that addition of disulfiramplus copper to the binding reaction reduces DNA binding to CRE. Lane 1,nuclear protein from fetal bovine serum-stimulated M1619 cells (FBS);lane 2, FBS+DMSO vehicle; lane 3, FBS+disulfiram (5 μM); lane 4, FBS+1.6μM CuSO₄; lane 5, FBS+disulfiram+CuSO₄; lane 6, FBS alone; lane 7,FBS+disulfiram; lane 8, FBS+CuSO_(4; lane) 9, FBS+disulfiram+CuSO₄; lane10, FBS+disulfiram+CuSO4. In lanes 1-5, DTT (2′.5 mM) was added to thebinding reaction as a reducing agent, whereas in lane 6-9, GSH (3.0 mM)was used. Disulfiram alone (lane 3) or disulfiram and copper (lane 5)reduced transcription factor binding to CRE, but the effect of theseagents was more pronounced when the binding reaction was performed withGSH (lanes 7 and 9) instead of DTT (lane 3 and 5) as the reducing agent.Inhibition of binding to CRE by disulfiram and copper in the presenceGSH was reversed by simultaneous addition of the more potent reducingagent DTT (lane 10).

The addition of disulfiram alone to the binding reaction reduced DNAbinding to CRE in the Upper ATF2 containing complex II (FIG. 2, lane 3).This effect was magnified when disulfiram was combined with copper ions(lane 5). These results are consistent width modest disruption of ATF2binding to CRE from formation of mixed disulfides between disulfiram andcysteines in the DNA binding region, and suggest that copper catalyzesmixed disulfide generation. However, reduction in CRE binding was muchmore pronounced when the binding reaction was performed with GSH insteadof DTT as the reducing agent (FIG. 2, lane 7 for disulfiram, lane 9 fordisulfiram plus copper). Inhibition of ATF2 containing complex IIbinding to CRE by disulfiram and copper in the presence of GSH wasreversed by simultaneous addition of the potent uncharged reducing agentDTT (FIG. 2, lane 10).

These results indicate that GSH, a cellular monothiol found in mMconcentrations within the nuclear compartment might react with thedithiocarbamate adduct leading to a bulky, negatively chargedGSH-containing mixed disulfide that could more effectively disrupt DNAbinding of ATF2.

EXAMPLE 2

This example shows that dithiocarbamate disulfides and copper inhibitcyclin A expression. It is known that heterodimers of the transcriptionfactors CREB-1 and c-Fos or ATF2 and Jun family members positivelyregulate cyclin-A expression through binding to a CRE element in thecyclin A promoter.

Since disulfiram and copper disrupt transcription factor DNA binding toCRE, their effect on expression of cyclin A was studied. FIG. 3A showsdisulfiram and copper reduce expression ofthe cell-cycle protein cyclinA. M1619 melanoma cells were plated at equal densities in 60×15 mmplastic dishes, grown to 80% confluence and treated with DMSO vehicle (5μl/ml), disulfiram (DS, 5 μM), or the combination of disulfiram andCuSO₄ (1.6 μM). After the indicated times, cells were lysed and proteinextracts were subjected to SDS-polyacrylamide gel electrophoresis (PAGE)followed by Western blotting using a rabbit polyclonal antibody (SantaCruz). Typical experiments are shown for 2, 4, 8, 12, 24 and 36 hours oftreatment with disulfiram plus CuSO₄.

FIG. 3B replicates experiments (n=4 each) in which cells were treatedwith DMSO vehicle, (5 μl/ml, lanes 1-4), disulfiram (5 μM, lanes 5-8),(5 μl/ml), CuSO₄ (1.6 μM, lanes 9-12) or the combination of disulfiramand CuSO₄ (lanes 13-16). After 24 hours cells were lysed, immunoblotswere performed to assay for cyclin A. In FIG. 3C shows quantitation ofexperiments in FIG. 3B by densitometry. Mean sum intensity of bands isdisplayed. *p<0.001 compared to all other treatments.

While disulfiram or copper alone had little effect (FIGS. 3B and C),treatment with the combination of disulfiram plus copper progressivelydecreased cyclin A expression over time (FIG. 3A) and reduced expressionof cyclin A by over two-thirds at 24 hours (FIGS. 3B and 3C). Incontrast, levels of B1 remained unchanged, and, in the cell lines westudied, disulfiram had no consistent effect on expression of the cellcycle inhibitor p21^(WAF1/CIP1) or the pro- and anti-apoptotic proteinsp53 or bcl-2 (data not shown).

EXAMPLE 3

This example illustrates that disulfiram is antiproliferative againstmelanoma and other tumor cell lines. Disruption of cyclin A expressionshould impair cell cycle progression and cellular proliferation.Therefore, the effect of disulfiram on M1619 melanoma growth, usingconcentrations readily achieved in humans on usual clinical doses wasstudied. Disulfiram was a potent inhibitor of growth in vitro for M1619melanoma (FIG. 4A). FIG. 4A shows that disulfiram inhibits proliferationof M1619 human melanoma cell lines. Cells stimulated with 10% fetalbovine serum (FBS) were plated at a density of 50,000 cells per well,and DMSO vehicle (5 μl per ml) or disulfiram (DS) was added to wells atthe indicated concentrations. After 24 hours, proliferation wasquantitated by assessing the cell number-dependent reduction of thesoluble yellow tetrazolium dye 3-[4,5-dimethylthiazol]-2yl-2,5-diphenyltetrazolium bromide (MTT) to its insoluble formazan, measured as theabsorbance at 540 nm (A₅₄₀) (6,7). *p<0.01 compared to FBS+DMSO vehiclecontrol.

Disulfiram also inhibited growth of a variety of other malignant celllines, including M1585 melanoma, prostatic adenocarcinoma, non-smallcell and small cell lung cancer, and adenocarcinoma of the breast (Table1). This was true whether disulfiram was added to culture Each valuerepresents mean±SE percent inhibition of growth comrpared to DMSOvehicle treated control cultures. Cells stimulated with 10% fetal bovineserum (FBS) were plated at a density of 50,000 cells per well. In somestudies (treatment initially) DMSO vehicle (5 μl per ml) or disulfiram(DS) was added to wells at the indicated concentrations. After 48 hours,proliferation was quantitated as described in FIG. 4.

In other studies (treatment after 24 hours) cells were grown for 24hours (M1619, M1585 and H596 lung) or 48 hours (breast). DMSO vehicle (5μl per ml) or disulfiram (DS) was added to wells at the indicatedconcentrations. After an additional 24 hours (lung) or 48 hours(breast), proliferation was quantitated as described in FIG. 4. Percentinhibition of growth was calculated as 100×(1.0−A₅₄₀ of MTT formazan indisulfiram treated cells/mean A₅₄₀ of MTT formazan in DMSO vehicletreated cells). In some cell lines, a modest (<10%) but statisticallysignificant inhibitory effect was observed with DMSO vehicle alone. Eachvalue represents a mean of at least 4 experiments. ^(A)p<0.01 comparedto FBS+DMSO vehicle control.

TABLE 1 DISULFIRAM IS ANTIPROLIFERATIVE FOR MALIGNANT CELLS Mean PercentInhibition of Growth Concentration of Disulfiram (μM) Cell Line 0.6251.25 2.5 5.0 Treatment initially Melanoma 100 ± 0^(A) 100 ± 0^(A) 100 ±0^(A) 100 ± 0^(A) M1585 Prostate  6 ± 6  29 ± 5^(A)  48 ± 2^(A)  86 ±2^(A) carcinoma CRL 1435 (PC-3) Squamous lung  76 ± 3^(A)  82 ± 4^(A) 77 ± 4^(A)  78 ± 3^(A) carcinoma NCI- H520 Adenosquamous  47 ± 4^(A) 57 ± 4^(A)  50 ± 3^(A)  50 ± 4^(A) lung carcinoma NCI-H596 Small celllung  68 ± 3^(A)  76 ± 6^(A)  76 ± 5^(A)  72 ± 3^(A) carcinoma NCI- H82Breast carcinoma  69 ± 4^(A)  94 ± 2^(A) 100 ± 0^(A) 100 ± 0^(A)MDA-MB-453 Treatment after 24 hours Melanoma  59 ± 4^(A)  35 ± 4^(A)  39± 3^(A)  37 ± 4^(A) M1619 Melanoma  74 ± 4^(A)  49 ± 7^(A)  41 ± 2^(A) 37 ± 6^(A) M1585 Lung carcinoma  30 ± 3^(A)  30 ± 3^(A)  29 ± 1^(A)  34± 3^(A) NCI-H596 Breast carcinoma  26 ± 5^(A)  26 ± 2^(A)  39 ± 2^(A) 46 ± 4^(A) MDA-MB-453

In FIG. 4B it is shown that the cell-impermeate Cu²⁺ chelatorbathocuproine-disulfonic acid prevents growth inhibition fromdisulfiram. M1619 melanoma cells stimulated and plated as described inA, and 1.25 μM disulfiram (DS) or DMSO vehicle (5 μl per ml) was addedto wells in the absence or presence of 50 or 100 μMbathocuproine-disulfonic acid (BCPS). After 48 hr proliferation wasquantitated as described. *p<0.001 compared to FBS+DMSO; +p<0.001compared to FBS+DS.

FIG. 4C shows that supplementation of growth medium with copper enhancesthe antiproliferative activity of disulfiram. M1619 melanoma cellsplated and stimulated as described in FIG. 4A were grown for 24 hoursand supplemented with CuSO₄ or CuSO₄ plus, 0.625 μM disulfiram. After anadditional 24 hours proliferation was quantitated. The addition of even0.2 μM CuSO₄ to medium converts 0.625 μM disulfiram from a 50%inhibitory (IC₅₀) concentration (A) into a 100% inhibitory (IC₁₀₀)concentration of drug. *p<0.001 compared to no CuSO₄;

The results shown in FIG. 4D illustrate that ceruloplasmin can serve asa source of copper for enhancing the antiproliferative activity ofdisulfiram. M1619 melanoma cells were plated, stimulated and grown for24 hours in the presence or absence of 0.625 μM disulfiram or 5 μl/mlDMSO vehicle in the presence or absence of human ceruloplasmin (Cerulo)at a concentration representing the upper level in normal human serum(500 μg/ml). After 24 hours proliferation was quantitated. *p<0.001compared to FBS+DMSO; +p<0.001 compared to FBS+DS.

Disulfiram induced both necrosis and apoptosis. Treatment of monolayerswith even low doses of disulfiram markedly increased trypan blue dyeuptake (6±2, 8±3.6 and 94±18 trypan blue positive cells per well,respectively, for untreated, DMSO vehicle treated or H520 lungadenosquamous carcinoma cells treated with 0.625 μM disulfiram; 12±0.9,16.5±2.1 and 93±12 trypan blue positive cells per well, respectively,for untreated, DMSO-treated or H82 small cell lung cancer cells treatedwith 0.625 μM disulfiram; p<0.001 compared to untreated or DMSO vehicletreated controls). Disulfiram also enhanced 3′-OH fluoresceinend-labeling of DNA fragments (FIGS. 5A, and SB) and DNA laddering onethidium bromide-stained agarose gels (data not shown). Consistent withits recently reported effects on P-glycoprotein mediated drug resistance(See, T. W. Loo, et al., “Blockage of drug resistance in vitro bydisulfiram, a drug used to treat alcoholism,” J. Natl. Cancer Inst.92:898-902 (2000)), disulfiram augmented the antiproliferative effect ofother antineoplastic agents on melanoma cells, a tumor notoriouslyresistant to chemotherapeutic drugs.

In FIG. 5A, M1619 melanoma cells treated with DMSO vehicle. In FIG. 5B,M1619 melanoma cells treated with 5 μM disulfiram. Disulfiram markedlyincreases 3′-OH fluorescein end-labeling of DNA fragments. Cells weregrown to confluence on 35 mm Petri dishes or on glass slides and treatedfor 15 hours with disulfiram or DMSO as vehicle. Apoptosis was studiedby terminal deoxynucleotidyl transferase (TdT) dependent 3′-OHfluorescein end-labeling of DNA fragments, using a Fluorescein-FragEL™DNA fragmentation detection kit (Oncogene Research Products, Cambridge,Mass.).

Table 2 shows that the combination of disulfiram and cisplatin ordisulfiram and carmustine is significantly more antiproliferativeagainst M1619 cells than cisplatin or carmustine alone:

TABLE 2 DISULFIRAM POTENTIATES THE ANTIPROLIFERATIVE ACTIVITY OFCHEMOTHERAPEUTIC AGENTS A540 of MTT Formazan A. Cisplatin (ng/ml) DMSOvehicle Disulfiram 2.5 μM    0 1.433 ± 0.038    1 1.739 ± 0.041 1.369 ±0.033^(B)   10 1.447 ± 0.047 1.221 ± 0.028   100 1.372 ± 0.052 1.183 ±0.038^(A) 1,000 1.381 ± 0.098 0.921 ± 0.027^(A) B. Carmustine (μM) DMSOvehicle Disulfiram 0.6 μM    0 0.104 ± 0.010    1 0.197 ± 0.004 0.042 ±0.003^(C)   10 0.152 ± 0.011 0.025 ± 0.002^(C)   100 0.020 ± 0.002 0.030± 0.023 1,000 0.003 ± 0.000 0.004 ± 0.000

In section A M1619 melanoma cells were cultured in 10% FBS and RPMI1640at a density of 50,000 cells/well plates. After 48 hours cisplatin and2.5 μM disulfiram or DMSO (5 μl per ml) were added to medium. After anadditional 24 hours, proliferation was quantitated. Each bar representsmean MTT formazan absorbance in a minimum of 4 experiments. ^(A)p<0.05compared to DMSO vehicle; ^(B)p<0.0 1 compared to DMSO vehcile.

In section B M1619 cells were cultured as above with addition ofcarmustine and 0.6 μM disulfiram or DMSO (5 μl per ml) to medium. After24 hours, proliferation was quantitated. Each bar represents mean MTTformazan absorbance in a minimum of 4 experiments. ^(C)p<0.001 comparedto DMSO vehicle.

Disulfiram was more potent as a growth inhibitor of neoplastic celllines than its sulfhydryl-clontaining relative PDTC. As an example, the50% inhibitor concentration (IC₅₀) against M1585 melanoma cells wasapproximately 1.25 μM for PDTC but was only 0.3 μM for disulfiram. Thissuggests that the active antiproliferative construct of thiocarbamatesnot likely the reduced thiol-containing monomeric form employedfrequently as an antioxidant.

EXAMPLE 4

The antiproliferative activity of dithiocarbamate disulfides depends oncomplexation with copper. PDTC induces apoptosis in normal thymocytesthat is mediated by complexation of copper from fetal bovine serum inthe medium and subsequent facilitation of copper transport into cells.Because inhibition of CRE DNA-binding by disulfiram was shown to becopper dependent in FIGS. 1A-1C and FIG. 2, the growth inhibition ofM1619 cells by disulfiram was studied to determine whether it wascontingent on its ability to complex with metals present in growthmedium. FIG. 4A shows that disulfiram combined with copper inducesS-phase cell cycle arrest in M1619 melanoma cells and apopotosis.Unsynchronized M1619 melanoma cells were grown in the presence of DMSOvehicle (A), 5 μM disulfiram (B), or 5 μM disulfiram plus 250 μg/mlceruloplasmin (Cerulo) as a source of copper (C). Twenty-four hourslater, cells were harvested and flow cytometric analysis was performed.The proportion of nuclei in each phase of the cell cycle (brackets) wasdetermined with MODFIT DNA analysis software. Disulfiram increases theportion of cells in S phase. The combination of disulfiram andceruloplasmin further increases the number of cells in S phase, preventsprogression into the G₂-M cell cycle and induces apoptosis.

Table 3 below shows that disulfiram greatly enhances intracellularuptake of copper, while FIG. 4B shows that the potent, cell impermeateCu²⁺ chelator bathocuproinie disulfonic acid (BCPS) greatly reducesgrowth inhibition from disulfiram. Conversely, the antiproliferativeactivity of disulfiram is greatly enhanced by supplementation of mediumwith concentrations of copper that do not by themselves affect cellgrowth (FIG. 4C). The copper transport protein ceruloplasmin, at levelsnormally present in human serum, can also serve as a source of copperthat can be complexed to enhance the antiproliferative activity ofdisulfiram (FIG. 4D).

Disulfiram treatment of M1619 melanoma cultures (FIG. 4B) slightlyreduces the number of cells in G₀-G₁ and increases the portion in Sphase of the cell cycle. The addition of copper from ceruloplasmin totreatment with disulfiram greatly magnifies these effects. Overtwo-thirds of cells are in S phase, none are in G₂-M, and 6% areapoptotic as identified by flow cytometric cell cycle analysis (FIG.4C). These studies suggest that growth inhibition of malignant celllines by dithiocarbamates and their disulfides is not only dependentupon interaction with certain metal ions, but also from complexationwith these metal ions and enhancing their intracellular transport.

TABLE 3 EFFECT OF DISULFIRAM ON INTRACELLULAR COPPER Treatment Copper(ng/ml) 10% FBS  56 ± 7 FBS + DMSO  52 ± 4 FBS + 0.625 μM DS  76 ± 11FBS + 1.25 μM DS 102 ± 5^(A) FBS + 2.5 μM DS 160 ± 17^(A) FBS + 5.0 μMDS 195 ± 3^(B)

M1619 melanoma cells were cultured at a density of 50,000 cells/well in24 well plates in the presence of 10% FBS and grown to confluence.Disulfiram or DMSO vehicle (5 μl/ml) was added at the concentrationsindicated, and cells were incubated an additional 6 hours. Supematantwas removed from cells and monolayers were washed twice with DPBS. Cellswere scraped into 1.0 ml 3 N HCL/10% trichloroacetic acid and hydrolyzedat 70° C. for 16 hours. After centrifugation at 600 g×10 min, copper wasmeasured using inductively coupled plasma emission spectroscopy atwavelengths of 324.754 and 224.700 nm. Replicates of four are reported.To minimize metal contamination, plastic ware rather than glass was usedin experiments, and double-distilled, deionized water was used for allaqueous media. ^(A)p<0.01 compared to DMSO control; ^(B)p<0.001 comparedto DMSO control.

EXAMPLE 5

This example shows dithiocarbate disulfides do not decreaseproliferation through redox mechanisms.

Disullfirain failed to deplete GSH in M1619 cells (228+18 for FBS alone;254+7 for DMSO vehicle control; 273±11 nmoles GSH/μg cell protein for 5μM disulfiram), and the combination of 5.0 μM disulfiram and 1.6 μMCuSO₄ even increased intracellular GSH (293±16 nmoles GSH/μg cellprotein; p<0.05 compared to FBS alone). Likewise, nelither disulfiram(0.625 to 5 μM), CuSO₄ (0.2-1.6 μM) nor the combination of 1.25 μMdisulfiram and 0.2 to 1.6 μM CuSO₄ caused measurable generation ofreactive oxygen species in M1619 cells, measured using theH₂O₂-sensitive intracellular probe 2′,7′-dichloroflurorescin. See, “J.A. Royall, et al., “Evaluation of 2′,7′-dichlorofluorescin anddihydrorhodamine 123 as fluorescent probes for intracellular H₂O₂ incultured endothelial cells,” Archiv. Biochem. Biophys. 302:348-355(1993). The baseline fluorescence of 1,431±23 units was not increased byany of the treatments.

In addition, the potent antioxidant probucol did not significantlyinhibit growth of any of our tumor cell lines (data not shown).Augmentation of intracellular copper might also increase levels of thereactive nitrogen species nitric oxide (NO) through Cu²⁺-mediateddecomposition of S-nitrosoglutathione and other nitrosothiols (See, D.R. Arnelle, et al., “Diethyl dithiocarbamate-induced decomposition ofS-nitrosothiols,” Nitric Oxide. Biol. and Chem. 1:56-64 (1997); M. P.Gordge, et al., “Copper chelation-induced reduction of the biologicalactivity of S-nitrosothiols,” Brit. J. Pharmacol. 114:1083-1089 (1995);A. C. F. Gorren, et al., “Decomposition of S-nitrosoglutathione in thepresence of copper ions and glutathione. Archiv. Biochem. Biophys,”330:219-2238 (1996)). NO, in turn, is believed to induce mitochondrialpermeability transition and produce other effects, leading to apoptosis(See, S. B. Hortelano, et al., “Nitric oxide induces apoptosis viatriggering mitochrondrial permeability transition. FEBS Lett,”410:373-377 (1997); Y. H. Shen, et al., “Nitric oxide induces andinhibits apoptosis through different pathways,” FEBS Lett. 433:125-131(1998)).

While the nitric oxide synthase inhibitor N6-nitro-L-arginine (LNAME)alone slightly enhanced cellular growth (23.7±2.3% increase; p<0.01compared to DMSO vehicle control), LNAME did not eliminate theantiproliferative effect of disulfiram (36.8±4.0% inhibition bydisulfiram alone vs 26.7±3.1% inhibition of growth in the presence ofdisulfiram plus LNAME; p<0.001 for each compared to DMSO vehicle controlbut not significantly different from each other). Finally, functioningas an antioxidant, PDTC has been postulated to interfere with growth ofcolorectal carcinoma in part by reducing expression of cyclooxygenase-2.See, r. Chinery, Nature Med, supra; R. Chinery, Cancer Res, supra.However, cyclooxygenase inhibitors failed to reduce growth in the celllines we studied (data not shown). Thus, taken together, these datasuggest that disulfiram does not appear to inhibit growth by adverselyaffecting the cellular redox state.

EXAMPLE 6

This example illustrates that metals other than copper can enhance theantiproliferaiive activity of dithiocarbamate disulfides. The absorptionof copper at both the intestinal and cellular level is blocked by zinccations, leading to the use of zinc acetate as the preferred treatmentfor Wilson's disease, the inherited disorder of copper overload.

High zinc concentrations in culture media affect copper uptake andtransport in differentiated human colon adenocarcinoma cells thereforeit was determined whether zinc supplementation of medium could inhibitthe antiproliferative activity of disulfiram, which appeared to becopper-dependent. Instead of reducing activity, zinc chloride alsosubstantially enhanced the antiproliferative potential of disulfiram(FIG. 7A). Dithiocarbamates actively complex copper but can chelateother metals (See, R. P. Bums, et al., “1,1-dithiolato complexes of thetransition elements,” Adv. Inorg. Chem. Radiochem. 23:211-280 (1980)),raising the possibility that the activity of disulfiram might also beenhanced by supplementation with a variety of metal salts.

FIGS. 7A-7D show that other metals also protentiate antiproliferativeactivity of disulfiram. FIG. 7A shows that zinc potentiates theantiproliferative activity of disulfiram. M1619 cells were stimulatedand plated as in FIG. 4. After 24 hours cells were treated, withindicated concentrations of zinc chloride (ZnCl₂) in the absence orpresence of 0.625 μM disulfiram. After an additional 24 hr, cell numberwas quantitated. *p<0.01 compared to no ZnCl₂; +p<0.001 compared to noZnCl₂.

FIG. 7B shows that not only copper and zinc, but also salts of gold andsilver can synergistically enhance the antiproliferative activity ofdisulfiram. This further supports the hypothesis that impairment ofcellular proliferation by disulfiram and possibly other dithiocarbamatesand their disulfides is dependent upon and enhanced catalytically by thepresence of heavy metals. In FIG. 7B the antiproliferative activity ofdisulfiram is enhanced by supplementation of medium with other heavymetals. M1619 cells plated and stimulated as above were treated with FBSalone, DMSO vehicle (5 μl/ml), disulfiram (DS, 0.15 μM), 5 μMconcentrations of metal salts (cupric sulfate, CuSO₄; silver lactate,C₃H₅AgO₃; gold chloride, HAuCl₄3H₂O) or the combination of DS plus metalsalts. After 48 hr cell number was quantitated. *p<0.05 compared toDMSO; +p<0.001 compared to DS alone.

In FIG. 7C complexes of disulfiram with gold demonstrate enhancedantiproliferative activity. M1619 cells plated and stimulated as abovewere treated with FBS alone, DMSO vehicle (5 μl/ml), disulfiram (DS, 160nM) or concentrations of gold complexed with disulfiram as outlined inMethods (AuDS). After 48 hr cell number was quantitated. *p<0.001compared to DMSO; +p<0.001 compared to DS.

EXAMPLE 7

This example shows thiolate anion formation mediates theantiproliferative activity of dithiocarbamates and their disulfides.

In light of the above findings with metals, chelates of disulfiram witha number of metal ions, including Cu²⁺, Zn²⁺, Ag¹⁺, or Au³⁺ weresynthesized. During generation of disulfiram-metal complexes, chelationof metal ions from the aqueous phase was suggested by a color change inthe disulfiram-containing chloroform phase (from pale yellow tobrilliant golden orange with complexation of gold ions). All metalcomplexes showed increased antiproliferative activity compared todisulfiram, but the most active compound was formed by the complex ofgold with disulfiram (FIG. 7C), which was antiproliferative at nMconcentrations.

The x-ray crystallographic structure of this compound revealed it to bea chelate of gold by the thiolate anion of diethyldiothiocarbamate, withchlorides occupying the other two valences of gold (FIG. 8). Complexeswere generated as outlined in Methods. Crystals were mounted on a NoniusKappa-CCD diffractometer for evaluation. The crystal diffracted well anda data set was collected to 27.5° in θ using Mo Kα radiation (λ0.7173A). Least-squares-refinement on the cell parameters reveled anorthorhombic P cell with unit cell parameters of a=11.5167(5),b=7.2472(2), c=12.9350(7) A, and a Volume of 1079.6(1) A³. Examinationof the systematic absences showed the space group to be Pnma. Thestructure was solved by direct methods using SIR₉₂ and revealed thecrystal to be dichloro(diethylthiocarbamyl)gold (II). The structure wasconfirmed by the successful solution and refinement of the 83independent variables for the 893 reflections (I>3δ(I)) to R-factors of3.3 and 3.2%, with an ESD of 1.499. The gold complex is a square planarcomplex in which the Au and the four coordinated atoms sit on a mirrorat x, 0.25, z. The organic ligand was found to be disordered with thediethylamine ligand occupying two sites related to each other throughthe mirror plane.

These results suggest that the proximate reactive dithiocarbamatestructure important for promoting cellular mixed disulfide formationmight be the thiolate anion generated from fully reduceddithiocarbamates or their disulfides by copper and other metals. To testthis hypothesis the ability of the thiolate sodiumdiethyldithiocarbamate to inhibit M1619 proliferation alone or in thepresence of a low concentration of DTT added to growth medium to promoteformation of the fully reduced thioacid was compared. FIG. 7D shows thatgrowth inhibition by the thiolate is greatly impaired by a concentrationof DTT that does not affect growth of melanoma cells alone. In FIG. 7Dthe antiproliferative activity of the thiolate sodiumdiethyldithiocarbamate trihydrate (NaDDC) is reduced by lowconcentrations of DTT in the growth medium. M1619 cells plated andstimulated above were treated with FBS alone, NaDDC (1 μM), DTT (100 μM)or NaDDC plus DTT. After 48 hours cell number was quantitated. *p<0.001compared to FBS; +p<0.001 compared to NaDDC alone. Thus, the function ofmetals in disrupting transcription factor DNA binding and cellproliferation may be to promote formation of the dithiocarbamate anion,the reactive chemical form that condenses into mixed disulfides with DNAbinding region cysteines, with secondary conjugation to GSH, effectingtranscription factor S-glutathionylation.

Many modification and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains, havingthe benefit of the teachings presented in the descriptions and theassociated drawings contained herein. Therefore, it is to be understoodthat the invention is not to be limited to the specific embodimentsdisclosed and that modifications and other embodiments are intended tobe included within the scope of the appended claims. Although specificterms are employed herein, they are used in a generic and descriptivesense only and not for purposes of limitation.

That which is claimed is:
 1. A method of treating cancer in human cellssensitive to a dithiocarbamate thiolate anion of the formula set forthbelow and for sensitizing tumors to conventional cancer chemotherapy byblocking the P-glycoprotein membrane toxin extrusion pump and forsensitizing AIDS patients to anti-retroviral therapy by blocking theP-glycoprotein membrane toxin extrusion pump comprising administering toa human subject in need thereof a therapeutically effective amount of adithiocarbamate thiolate anion of the formula:

wherein R₂ and R₃ are the same or different and represent hydrogen, andunsubstituted or substituted alkyl, akenyl, aryl, and alkoxy groups; Mis an alkali metal selected from the group consisting of sodium,potassium, calcium, magnesium, barium, and lithium; An is copper; n isthe valence of the metal.
 2. The method according to claim 1 whereinsaid dithiocarbamate thiolate anion is in the form of a pharmaceuticallyacceptable salt.
 3. The method according to claim 1 wherein saiddithiocarbamate thiolate anion is administered in a dosage of betweenabout 125 to about 1000 mg per day of body weight.
 4. The methodaccording to claim 1 wherein said dithiocarbamate thiolate anion isadministered in a dosage of between about 250 to about 500 mg per day.5. The method according to claim 1 wherein said dithiocarbamate thiolateanion is administered parenterally.
 6. The method according to claim 1wherein said dithiocarbamate thiolate anion is administered orally. 7.The method according to claim 1 wherein said cancer is melanoma, lungcancer, renal cancer, colorectal cancer, breast cancer, pancreaticcancer, gastric cancer, bladder cancer, ovarian cancer, uterine cancer,lymphoma, and prostrate cancer.
 8. The method according to claim 1wherein said alkyl, akenyl, aryl and alkoxy groups are substituted. 9.The method according to claim 1 wherein said aryl group is a heteroaryl.